Saltatory rolling circle amplification (SRCA) combined with Safranine O for the rapid, sensitive and visual detection of Listeria monocytogenes in food

Abstract

Listeria monocytogenes (L. monocytogenes) is one of the most prevalent foodborne pathogens. It causes intestinal infections as well as severe meningitis and septicemia, representing a significant risk to food safety and human health. Consequently, the present study proposes a novel method based on the combination of saltatory rolling circle amplification (SRCA) technology and Safranine O for the rapid, sensitive and visual detection of L. monocytogenes in food. In the presence of L. monocytogenes, the SRCA amplification reaction yields copious quantities of double-stranded DNA. Following the addition of Safranine O and the oxidant, Safranine O is oxidized to form Safranine O oligomer. The electrostatic interactions between the positively charged Safranine O oligomers and the negatively charged SRCA amplification product result in the formation of dark red visible aggregates, the absorbance value also decreases with the formation of aggregates. The method has low detection limit, high specificity, straightforward and time efficient (DNA amplification for 40 min, aggregation for 10 min). The visual qualitative detection limit was 2.7 × 10¹ CFU/mL, and the limit of detection quantitative detection was 3.4 CFU/mL. In comparison with the SRCA electrophoresis detection method, the visual detection limit was enhanced by 10 times. Comparison with ISO method, the sensitivity, specificity and accuracy of the method for the detection of real samples were 100%, 96.2% and 96.7%, respectively. This method offers a straightforward and accurate qualitative and quantitative approach for the identification of L. monocytogenes in food, and is particularly suited to the detection of pathogenic bacteria in food in resource-poor areas.